ABSTRACTPTEN-Long is a translational variant of PTEN (Phosphatase and Tensin Homolog). Like PTEN, PTEN-Long is able to antagonize the PI3K-Akt pathway and inhibits tumor growth. In this study, we investigated the role PTEN-Long plays in the development and progression of clear cell renal cell carcinoma (ccRCC) and explored the therapeutic possibility using proteinaceous PTEN-Long to treat ccRCC. We found that the protein levels of PTEN-Long were drastically reduced in ccRCC, which was correlated with increased levels of phosphorylated Akt (pAkt). Gain of function experiments showed overexpression of PTEN-Long in the ccRCC cell line 786-0 suppressed PI3K-Akt signaling, inhibited cell proliferation, migration and invasion, and eventually induced cell death. When purified PTEN-Long was added into cultured 786-0 cells, it entered cells, blocked Akt activation, and induced apoptosis involving Caspase 3 cleavage. Furthermore, PTEN-Long inhibited proliferation of 786-0 cells in xenograft mouse model. Our results implicated that understanding the roles of PTEN-Long in renal cell carcinogenesis has therapeutic significance.

pone-0114250-g005: PTEN-Long treatment in culture inhibits PI3K signal and induces apoptosis in 786-0 cells.A, Coomassie stained gel showing representative protein preparations of PTEN, PTENG129R, PTEN-Long, and PTEN-LongG302R. B, Effect of treatment with 25 nM PTEN-Long or other related proteins for 1h on pAkt and pPRAS40. PTEN-Long but not other proteins reduced intracellular phosphorylation of the Akt and PRAS40 in 786-0 cells. C and D, Effect of treatment with PTEN-Long or related proteins at different doses for 24 h on serum-starved 786-0 cells showed that PTEN-Long but not PTEN suppressed PI3K signaling and induced apoptosis, indicated by cleavage of caspase 3 in a dose-dependent manner. Percentages of apoptotic cells were also determined by PI/FACS (C). n = 4; bars represent the means ± SD. E, Representative pictures of the treated cells were taken by phase contrast photography. Protein concentrations were 50 nM. The scale bar represents 20 µm.

Mentions:
As PTEN-Long is a potential protein-type drug for cancer therapy [24], we next tested whether PTEN-Long is able to affect cellular signaling as an exogenous agent. Recombinant PTEN-Long, PTEN-LongG302R, PTEN and PTENG129R were expressed in bacteria and purified. The effects of proteins on cells were evaluated by direct application of purified products into cultured cells followed by Western blotting analysis of cell lysates prepared. The results showed that the only treatment of 786-0 cells in culture for 1 hr with purified PTEN-Long, but not other proteins, reduced intracellular phosphorylation of the Akt and PRAS40 in 786-0 cells (Fig. 5A and 5B). Moreover, dose-response experiments with 786-0 cells deprived of serum and treatment for 24 h showed that PTEN-Long both suppressed PI3K signaling, which was indicated by reduced levels of phosphorylated Akt and PRAS40, and also induced cell apoptosis, indicated by cleavage of caspase 3 in a dose-dependent manner (Fig. 5C) and increased percentages of sub G0/G1 population determined by PI/FACS (Fig. 5D). In comparison, no effect was seen from treatment of cells with purified PTEN at various doses (Fig. 5C–5E), suggesting PTEN-Long has therapeutic advantage over PTEN.

pone-0114250-g005: PTEN-Long treatment in culture inhibits PI3K signal and induces apoptosis in 786-0 cells.A, Coomassie stained gel showing representative protein preparations of PTEN, PTENG129R, PTEN-Long, and PTEN-LongG302R. B, Effect of treatment with 25 nM PTEN-Long or other related proteins for 1h on pAkt and pPRAS40. PTEN-Long but not other proteins reduced intracellular phosphorylation of the Akt and PRAS40 in 786-0 cells. C and D, Effect of treatment with PTEN-Long or related proteins at different doses for 24 h on serum-starved 786-0 cells showed that PTEN-Long but not PTEN suppressed PI3K signaling and induced apoptosis, indicated by cleavage of caspase 3 in a dose-dependent manner. Percentages of apoptotic cells were also determined by PI/FACS (C). n = 4; bars represent the means ± SD. E, Representative pictures of the treated cells were taken by phase contrast photography. Protein concentrations were 50 nM. The scale bar represents 20 µm.

Mentions:
As PTEN-Long is a potential protein-type drug for cancer therapy [24], we next tested whether PTEN-Long is able to affect cellular signaling as an exogenous agent. Recombinant PTEN-Long, PTEN-LongG302R, PTEN and PTENG129R were expressed in bacteria and purified. The effects of proteins on cells were evaluated by direct application of purified products into cultured cells followed by Western blotting analysis of cell lysates prepared. The results showed that the only treatment of 786-0 cells in culture for 1 hr with purified PTEN-Long, but not other proteins, reduced intracellular phosphorylation of the Akt and PRAS40 in 786-0 cells (Fig. 5A and 5B). Moreover, dose-response experiments with 786-0 cells deprived of serum and treatment for 24 h showed that PTEN-Long both suppressed PI3K signaling, which was indicated by reduced levels of phosphorylated Akt and PRAS40, and also induced cell apoptosis, indicated by cleavage of caspase 3 in a dose-dependent manner (Fig. 5C) and increased percentages of sub G0/G1 population determined by PI/FACS (Fig. 5D). In comparison, no effect was seen from treatment of cells with purified PTEN at various doses (Fig. 5C–5E), suggesting PTEN-Long has therapeutic advantage over PTEN.

Bottom Line:
We found that the protein levels of PTEN-Long were drastically reduced in ccRCC, which was correlated with increased levels of phosphorylated Akt (pAkt).When purified PTEN-Long was added into cultured 786-0 cells, it entered cells, blocked Akt activation, and induced apoptosis involving Caspase 3 cleavage.Furthermore, PTEN-Long inhibited proliferation of 786-0 cells in xenograft mouse model.

ABSTRACTPTEN-Long is a translational variant of PTEN (Phosphatase and Tensin Homolog). Like PTEN, PTEN-Long is able to antagonize the PI3K-Akt pathway and inhibits tumor growth. In this study, we investigated the role PTEN-Long plays in the development and progression of clear cell renal cell carcinoma (ccRCC) and explored the therapeutic possibility using proteinaceous PTEN-Long to treat ccRCC. We found that the protein levels of PTEN-Long were drastically reduced in ccRCC, which was correlated with increased levels of phosphorylated Akt (pAkt). Gain of function experiments showed overexpression of PTEN-Long in the ccRCC cell line 786-0 suppressed PI3K-Akt signaling, inhibited cell proliferation, migration and invasion, and eventually induced cell death. When purified PTEN-Long was added into cultured 786-0 cells, it entered cells, blocked Akt activation, and induced apoptosis involving Caspase 3 cleavage. Furthermore, PTEN-Long inhibited proliferation of 786-0 cells in xenograft mouse model. Our results implicated that understanding the roles of PTEN-Long in renal cell carcinogenesis has therapeutic significance.